Objective:To establish and validate a fluorescence PCR with internal positive control for rapid Mycoplasma detection. Methods:A fluorescence PCR with internal positive control for Mycoplasma detection was developed and verified for its specificity, limit of detection, and robustness. A sample of fever with thrombocytopenia syndrome (SFTSV) virus strains was tested with this method, and the result was compared with those of culture method and indicator cell culture method. Results:The established fluorescence PCR had good specificity and could amplify 11 kinds of plasmids containing Mycoplasma 16S rRNA gene with high efficiency. There was no cross reaction with the genomic DNAs of Clostridium sporogenes, Clostridium acetobutylicum, Lactobacillus acidophilus, Streptococcus pneumoniae, Bacillus subtilis, Staphylococcus aureus, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans, Vero cells, RD cells, and SF9 cells. The amplification efficiency of the internal positive control was basically consistent with that of the target gene of Mycoplasma, suggesting that the internal positive control could be used to detect the presence of PCR inhibitors. The sensitivity of the established method was high, and the detection limit was 10 colony-forming unit (CFU)/ml for Mycoplasma fermentans, 5 CFU/ml for Mycoplasma arginine, 5 CFU/ml for Mycoplasma gallisepticum, 5 CFU/ml for Mycoplasma hyorhinis, 5 CFU/ml for Acholeplasma laidlawii, 5 CFU/ml for Mycoplasma orale, 5 CFU/ml for Mycoplasma pneumoniae, 5 CFU/ml for Mycoplasma synoviae, and 1 CFU/ml for Spiroplasma citri by 7500 Fast real-time PCR system. At the detection limit of each species, there was no significant difference in the positive detection rate using different thermal cycler types. The established fluorescence PCR, culture method, and indicator cell culture were performed to detect Mycoplasma in the sample of SFTSV virus strains, and the results all showed Mycoplasma contamination. Conclusions:The established fluorescence PCR has high specificity, sensitivity, and robustness, and can be used as an alternative method for rapid detection of Mycoplasma.

Objective To explore prescription medication rules and potential mechanism of traditional Chinese medicine(TCM)in the treatment of alcoholic liver disease(ALD)based on the technology of data mining and network pharmacology.Methods The prescriptions related to the treatment of ALD were retrieved in Chinese National Knowledge Infrastructure,Wanfang,Chinese Biomedical Literature and VIP databases.After the data were collated according to the filter criteria,IBM SPSS Statistics 27.0 and IBM SPSS Modeler 18 software were used to analyze the prescription rules and association rules.Then,the medication rules of TCM in the treatment of ALD were summarized,and the core drug combinations were obtained.Active ingredients in the core drug combinations for ALD and their targets were screened by network pharmacology.GO and KEGG analysis were performed on the main targets,and molecular docking technique was used to verify the binding ability of active ingredients to main targets.Results A total of 143 prescription for ALD were screened,involving 222 Chinese medicine,among which 28 high-frequency Chinese medicine were used with a frequency≥25 times.Eight core drug combinations were obtained by associations rule analysis.It has been found that there are 215 intersection targets between"Poria-Atractylodis macrocephalae Rhizoma-Hearba Artemisiae Scopariae"and ALD,including six core targets of AKT1,TNF,VEGFA,IL-1β,SRC,EGFR.One hundred and sixty-eight of signaling pathways are involved,including cancer pathways,PI3K/AKT signaling pathways,chemical carcinogenesis-reactive oxygen species,lipid and atherosclerosis,etc.Molecular docking results showed that the main active components including cerevisterol,genkwanin and demethoxycapillarisin had good binding ability to AKT1.Conclusion The main active ingredients in"Poria-Atractylodis macrocephalae Rhizoma-Hearba Artemisiae Scopariae"can participate in the regulation of key signaling pathways such as PI3K/AKT by acting on key target proteins(AKT1,TNF,and VEGFA).Subsequently,they play a role in inhibiting inflammatory response and apoptosis,slowing down liver fibrosis,and promoting hepatocyte repair.This study provides data support and theoretical guidance for the study of TCM in the treatment of ALD.

OBJECTIVES: Our previous study has verified that high level of SET and MYND domain-containing protein 2 (SMYD2) plays an important role in acquiring aggressive ability for liver cancer cells in hepatocellular carcinoma. MiR-200b as a tumor suppressor gene involves in a variety of cancers. This study aims to investigate the correlation between miR-200b and SMYD2 in hepatocellular carcinoma and the underlying mechanism. METHODS: Firstly, the levels of SMYD2 and miR-200b in hepatocellular carcinoma tissues and matched adjacent non-tumor liver tissues were tested with real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Secondly, we evaluated the interaction between miR-200b and SMYD2 using dual-luciferase reporter assay. Thirdly, we elucidated the effect of miR-200b on SMYD2 and its downstream targets p53/CyclinE1. Finally, we silenced SMYD2 in hepatocellular carcinoma cell lines to investigate its effect on tumor proliferation and cell cycle progression, and further confirmed the correlation among SMYD2 and p53/CyclinE1. RESULTS: Compared with the matched adjacent non-tumor liver tissues, miR-200b was obviously decreased, and SMYD2 was significantly increased in hepatocellular carcinoma (both P<0.05). Spearman's rank correlation revealed that miR-200b expression was negatively correlated with SMYD2 (P<0.01). Computer algorithm and dual-luciferase reporter assay revealed that miR-200b directly targeted and suppressed SMYD2 in HEK 293T cells. The down-regulated miR-200b expression promoted hepatoma cell proliferation (P<0.05) and increased SMYD2 expression(P<0.01), while the up-regulated expression of miR-200b had an opposite effect. The knockdown of SMYD2 suppressed the proliferation of MHCC-97L cells (P<0.01), down-regulated CyclinE1, and up-regulated p53 expression (both P<0.05). CONCLUSIONS MiR-200b is involved in hepatocellular carcinoma progression via targeting SMYD2 and regulating SMYD2/p53/CyclinE1 signaling pathway and may be used as a potential target for hepatocellular carcinoma treatment.
Humans ; Carcinoma, Hepatocellular/pathology* ; Tumor Suppressor Protein p53/metabolism* ; MicroRNAs/metabolism* ; Cell Line, Tumor ; Signal Transduction ; Liver Neoplasms/pathology* ; Cell Proliferation/genetics* ; Histone-Lysine N-Methyltransferase/metabolism*

OBJECTIVETacrolimus (FK506) is an immunosuppressive drug, which is widely used to prevent rejection of transplanted organs. However, chronic administration of FK506 leads to hypertension in solid organ transplantation patients, and its molecular mechanisms are much more complicated. In this review, we will discuss the above-mentioned molecular mechanisms of FK506-induced hypertension in solid organ transplantation subjects.

DATA SOURCESThe data analyzed in this review were mainly from relevant articles without restriction on the publication date reported in PubMed. The terms "FK506" or "tacrolimus" and "hypertension" were used for the literature search.

STUDY SELECTIONOriginal articles with no limitation of research design and critical reviews containing data relevant to FK506-induced hypertension and its molecular mechanisms were retrieved, reviewed and analyzed.

RESULTSThere are several molecular mechanisms attributed to FK506-induced hypertension in solid organ transplantation subjects. First, FK506 binds FK506 binding protein 12 and its related isoform 12.6 (FKBP12/12.6) and removes them from intracellular ryanodine receptors that induce a calcium ion leakage from the endoplasmic/sarcoplasmic reticulum. The conventional protein kinase C beta II (cPKCβII)-mediated phosphorylation of endothelial nitric oxide (NO) synthase at Thr495, which reduces the production of NO, was activated by calcium ion leakage. Second, transforming growth factor receptor/SMAD2/3 signaling activation plays an important role in Treg/Th17 cell imbalance in T cells which toget converge to cause inflammation, endothelial dysfunction, and hypertension following tacrolimus treatment. Third, the activation of with-no-K(Lys) kinases/STE20/SPS1-related proline/alanine-rich kinase/thiazide-sensitive sodium chloride co-transporter (WNKs/SPAK/NCC) pathway has a central role in tacrolimus-induced hypertension. Finally, the enhanced activity of renal renin-angiotensin-aldosterone system seems to play a crucial role in the pathophysiology of FK506-induced hypertension.

CONCLUSIONFK506 plays a predominant role in the pathophysiology of hypertension in solid organ transplantation subjects.

Humans ; Hypertension ; chemically induced ; Immunosuppressive Agents ; adverse effects ; therapeutic use ; Organ Transplantation ; adverse effects ; Tacrolimus ; adverse effects ; therapeutic use

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3β and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.
Animals ; Bone Morphogenetic Protein 4/metabolism* ; Cell Differentiation ; Chromosomal Instability ; Endosomal Sorting Complexes Required for Transport ; Mice ; Mitogen-Activated Protein Kinase Kinases/metabolism* ; Mouse Embryonic Stem Cells/cytology* ; Pluripotent Stem Cells/cytology* ; Signal Transduction ; Ubiquitin-Conjugating Enzymes